Pierce Silver Stain Kit
Wash gel in ultra pure water for 5 minutes .
Replace the water and wash for another 5 minutes .
Fix gel in 30 % ethanol : 10 % acetic acid solution ( i . e . , 6 : 3 : 1 water : ethanol : acetic acid ) for 15 minutes .
Replace the solution and fix for another 15 minutes .
Note : Gel may be kept in fixing solution overnight without affecting stain performance .
Wash gel in 10 % ethanol solution for 5 minutes .
Replace solution and wash for another 5 minutes .
Wash gel in ultrapure water for 5 minutes .
Replace water and wash for another 5 minutes .
Prepare Sensitizer Working Solution by mixing 1 part Silver Stain Sensitizer with 500 parts ultrapure water ( e . g . , mix 50µL Sensitizer with 25mL water ) .
Incubate gel in Sensitizer Working Solution for exactly 1 minute , then wash with two changes of ultrapure water for 1 minute each .
Prepare Stain Working Solution by mixing 1 part Silver Stain Enhancer with 50 parts Silver Stain ( e . g . , 0.5mL of Enhancer with 25mL Stain ) .
Incubate gel in Stain Working Solution for 30 minutes .
Note : Gel may be incubated in Stain Working Solution for as short as 5 minutes or as long as overnight without affecting stain performance .
Prepare Developer Working Solution by mixing 1 part Silver Stain Enhancer with 50 parts Silver Stain Developer ( e . g . , mix 0.5mL of Enhancer with 25mL Developer ) .
Prepare 5 % acetic acid solution as a Stop Solution .
Quickly wash gel with two changes of ultrapure water for 20 seconds each .
Immediately add Developer Working Solution and incubate until protein bands appear ( 2 - 3 minutes ) .
Note : Protein bands will begin to appear within 30 seconds and then continue to develop .
Between 2 and 3 minutes , protein detection vs . background is optimal .
After 3 minutes , lane background signal may increase to undesirable levels .
When the desired band intensity is reached , replace Developer Working Solution with prepared Stop Solution ( 5 % acetic acid ) .
Wash gel briefly , then replace Stop Solution and incubate for 10 minutes .
